GENE STRUCTURE, ACTIVITY AND LOCALIZATION OF A CATALASE FROM INTRACELLULAR BACTERIA IN ONCHOCERCA-VOLVULUS

Within the context of studies on the antioxidant enzymes in Onchocerca volvulus, DNA clones encoding catalase (CAT) were isolated from an O. volvulus adult lambda zapII cDNA library. Analysis of their nucleotid e and encoded amino acid sequences revealed that they derive from intr acellular bacteria, rather than the O. volvulus nuclear genome. The en dobacterial CAT gene was found to lie in a gene cluster, followed by a ferritin gene and an excinuclease gene. The endobacterial CAT gene en codes a functional enzyme capable of detoxifying H2O2, demonstrated by producing an active recombinant protein in an E. coli expression syst em. The purified 54 kDa protein has CAT activity over a broad pH range , with a specific activity of 103000 +/- 3000 U mg(-1). The optical sp ectrum of the endobacterial CAT shows that it is a ferric haem-contain ing protein with a Soret band at 405 Mm. To investigate the phylogeny of the intracellular bacterium in O. volvulus, a segment of the 16S rR NA gene was amplified from total genomic DNA by a polymerase chain rea ction using universal eubacterial primers. A phylogenetic analysis of the O. volvulus-derived 16S rRNA sequence revealed that the endobacter ium belongs to a distinct Wolbachia clade of the order Rickettsiales. Onchocercomata and biopsies containing different onchocercal species w ere immunohistochemically stained using polyclonal antibodies raised a gainst the recombinant endobacterial CAT. CAT was detected in the endo bacteria in the hypodermis of adult male and female O. volvulus, O. oc hengi, O. gibsoni and O. fasciata. The endobacterial enzyme was also d etected in onchocercal oocytes and all embryonic stages including intr auterine microfilariae as well. as skin microfilariae. O. volvulus thu s harbours Wolbachia-like endosymbionts which are transovarially trans mitted and show particular affinity for the hypodermal tissues of the lateral chords. (C) 1998 Elsevier Science B.V. All rights reserved.